Anthrax toxin protective antigen protein (PA, 83 kDa) binds to receptors on the surface of mammalian cells, is cleaved by the cell surface protease furin, and then captures either of the two other toxin proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocytosis via lipid rafts and pass through endocytic vesicle populations, and then translocate LF and EF to the cytosol. EF is a calcium and calmodulin-dependent adenylyl cyclase that causes large and unregulated increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs). The toxin proteins play a central role in the virulence of Bacillus anthracis. The PA protein is the key ingredient in anthrax vaccines. The toxin can be modified to have specificity for tumor cells and is being developed as an anti-cancer agent. [unreadable] [unreadable] Progress was achieved in a number of areas, including the following. (1) In continuation of structure-function studies, mutated PA proteins were obtained by bacteriophage display that discriminate between the two cell surface receptors, TEM8 and CMG2. These proteins will help delineate the importance of each receptor in determining the sensitivity of various tissues and organs to the toxin. Methods for production of the toxin proteins were improved and the technologies for this were transferred to several laboratories. In separate studies, cells expressing recombinant forms of either CMG2 or TEM8 were used to show that the latter receptor has a 3-fold lower affinity for PA and that this explains in part the observation that TEM8, unlike CMG2, allows toxin translocation to the cytosol from early endosomes. (2) Thorough analysis of the fate and processing of PA in the blood of mice and rats has helped to explain the action of the toxins in animals. It was confirmed that PA is cleaved in the blood of mice, but no cleavage occurs in rats. When present in large amounts, the cleaved PA can bind LF and decrease its effects. Cleavage of PA in the blood or on cells promotes its uptake by cells and its removal from the blood. These observations helped to explain the inhibitory actions of several mutated PA proteins. (3) The collaborative project with the NHGRI Chemical Genomics group using the fusion protein containing the N-terminal portion of LF and the enzyme beta-lactamase confirmed the validity of the approach by generating a number of candidate toxin inhibitors. These are undergoing detailed analysis. (4) It was demonstrated that the edema toxin (PA+EF) sensitizes DBA/2J mice to the action of the lethal toxin (PA+LF), possibly through its action on adrenal glands. This finding is one of the first to show that the two toxins act synergistically in animals. (5) Collaborative studies with two laboratories on the in vivo physiological effects of the edema and lethal toxin in mice and rats identified aspects of the shock response that may be informative in designing therapies. In particular, alterations in cardiac function and resulting hemodynamic dysfunctions were identified. (6) Collaborative work continued with several groups developing vaccines and therapeutics for anthrax. We worked with groups developing inhibitors of the anthrax lethal factor protease activity, human and humanized antibodies that neutralize anthrax toxin, and new methods for producing and delivering candidate vaccine antigens derived from the toxin and the poly-glutamic acid capsule. In one example, we collaborated in work that expressed various forms of PA, LF, and EF on the surface of bacteriophage and that tested these and other vaccine formulations administered by the transcutaneous route. Work with another group has progressed to a phase I clinical trial performed at NIH and other centers in which a mutated recombinant PA is being evaluated as a second generation human vaccine. (5) Knowledge of bacterial toxin structure and function was used to design cytotoxins specific for cancer cells. Ongoing studies on treating mouse tumors show that anthrax toxin fusion proteins work not only when injected adjacent to the tumor, but also when administered systemically, as would be preferred if these drugs were to be developed for human use. Furthermore, the native LF protein was shown to have good efficacy against all solid tumor types tested when used with a metalloproteinase-activated PA. This wide efficacy results from targeting of tumor vasculature rather than only the tumor cells.